Abstract

CD4+CD25+ regulatory T cells (Tregs) mediate peripheral T-cell homeostasis and contribute to self-tolerance. Their homeostatic and pathologic trafficking is poorly understood. Under homeostatic conditions, we show a relatively high prevalence of functional Tregs in human bone marrow. Bone marrow strongly expresses functional stromal-derived factor (CXCL12), the ligand for CXCR4. Human Tregs traffic to and are retained in bone marrow through CXCR4/CXCL12 signals as shown in chimeric nonobese diabetic/severe combined immunodeficient mice. Granulocyte colony-stimulating factor (G-CSF) reduces human bone marrow CXCL12 expression in vivo, associated with mobilization of marrow Tregs to peripheral blood in human volunteers. These findings show a mechanism for homeostatic Treg trafficking and indicate that bone marrow is a significant reservoir for Tregs. These data also suggest a novel mechanism explaining reduced acute graft-versus-host disease and improvement in autoimmune diseases following G-CSF treatment.

INTRODUCTION

CD4+CD25+ T cells (Tregs) mediate peripheral T-cell homeostasis
(1,
2,
3)
. Studies in murine models show that CD4+CD25+ T cells are essential for the induction of tolerance to alloantigens and inhibit graft-versus-host disease (GVHD; refs.
4
,
5
). Human blood Tregs express CCR4 and CCR8 and migrate in response to their ligands in vitro(6)
. We recently showed that pathologic Tregs in human ovarian cancers migrate into tumor in response to CCL22
(7)
. Homeostatic Tregs may originate in the thymus, although their differentiation in the periphery also has been suggested. Despite these recent advances in our knowledge, relatively little is known regarding the natural reservoirs of migrating Tregs or signals that induce their mobilization of trafficking.

Bone marrow is vascularized by blood but not by lymphatic vessels. Bone marrow is a part of the lymphocyte recirculation network
(8)
, with billions of lymphocytes recirculating through it each day. We hypothesized that bone marrow might harbor CD4+CD25+ Tregs and function as a reservoir for them. In this capacity, it could be an important organ to fine tune T-cell immunity by modulating Treg trafficking. We further hypothesized that granulocyte colony-stimulating factor (G-CSF) would mobilize bone marrow CD4+CD25+ Tregs. We tested these hypotheses in defined models and human subjects and now show a high prevalence of functional Tregs in human bone marrow. We provide evidence that CXCR4/CXCL12 signals play an important role in regulating Treg egress from bone marrow and in maintaining homeostatic levels of Tregs in the periphery. G-CSF mobilizes Tregs from bone marrow into the periphery by decreasing marrow CXCL12 expression.

These data may be used to manipulate Tregs for therapeutic purposes and may help to explain the low prevalence of acute GVHD in recipients of G-CSF–mobilized bone marrow transplants
(9, 10)
and the improvement in autoimmune diseases following G-CSF treatment
(11, 12)
.

MATERIALS AND METHODS

Human Subjects.

Written, informed consent was obtained for all of the subjects. The Institutional Review Board of Tulane Medical School approved the study. Healthy adults were studied untreated or immediately following subcutaneous injection of 5 μg/kg recombinant human G-CSF (Filgastrim; Amgen Inc., Thousand Oaks, CA) once daily for four consecutive days. Peripheral blood cells, bone marrow cells, and bone marrow fluid (cell-free bone marrow) were collected and frozen for later use. Human thymus and tonsils were collected from young children undergoing cardiac surgery or other treatments and mechanically disrupted into single cell suspensions. Cells were stained with monoclonal antibodies and analyzed on a FACSCalibur (Becton-Dickinson, Franklin Lakes, NJ) using mouse antihuman CD4-FITC (SK3, IgG1) and mouse antihuman CD25-phycoerythrin (MA251, IgG1; all from BD PharMingen, San Diego, CA).

Mice.

The study was approved by the Institutional Animal Care and Use Committee of the Tulane Medical School. Six- to 8-week-old female C57/black 6 mice were used (Jackson Labs, Bar Harbor, ME). Peripheral blood was collected by cardiac aspiration into heparinized glass tubes. Spleen and inguinal lymph nodes were mechanically disrupted into single cell suspensions. Bone marrow was collected from the femur, tibia, and humerus by flushing with 200 μL sterile PBS. After centrifugation, the supernatant (bone marrow fluid) was frozen for later detection of chemokines and for migration assays. CD3+CD4+CD25+ T cells in blood, spleen, and lymph nodes were identified with fluorescence-activated cell sorting (FACS) using antimouse CD4 (clone RM 4–5; eBioscience, San Diego, CA), CD25 (PC61; eBioscience), and CD3 (145–2c11; BD PharMingen). At least 5000 gated events per condition were analyzed using CellQuest software (Becton-Dickinson).

Reverse Transcription-PCR.

Real-time reverse transcription-PCR was carried out for FOXP3 using primers for upstream 5′-cagctgcccacactgcccctag-3′, downstream 5′-catttgccagcagtgggtag-3′, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). cDNA was against normalized GAPDH and expressed as fold difference relative to GAPDH(13)
.

Having shown a role for CXCL12/CXCR4 signals in vitro, we next addressed the role for this axis in vivo in our human–nonobese diabetic/severe combined immunodeficient (NOD/SCID) chimeric mouse model
(16)
. Forty to 60 hours after intravenous human Treg transfusion, human Tregs were primarily found in bone marrow. Far fewer Tregs were found in spleen (Fig. 4A)
⇓
, and <100 cells/mL were detected in peripheral blood. Strikingly, in vivo administration of a specific antihuman CXCR4 monoclonal antibody significantly (P < 0.05) decreased Treg migration into bone marrow but not into spleen (Fig. 4B)
⇓
. We further showed that mouse bone marrow produced high level of CXCL12 but not CCL22 or CCL17 (Fig. 4C)
⇓
. In further support, mouse bone marrow efficiently mediated human MDC-activated Treg migration in vitro in a dose-dependent manner. This in vitro migration was significantly blocked by mouse antihuman CXCR4 monoclonal antibody (Fig. 4D)
⇓
. Collectively, these data indicate that CXCL12/CXCR4 signals are critical for Treg trafficking to bone marrow in vivo.

DISCUSSION

Homeostatic Tregs mediate peripheral tolerance to self-antigens by suppressing autoreactive immune cells. Emerging evidence implicates regulatory T cells, particularly CD4+CD25+ regulatory T cells (Tregs) in the pathogenesis of autoimmune diseases, tumors, and organ transplantation
(2, 3, 17, 18, 26)
. Classic, natural CD4+CD25+ regulatory T cells are thought to reside primarily in lymphoid organs
(2, 3, 17, 18)
. On the basis of these observations, it is postulated that Tregs mediate their suppressive effects by inhibiting T-cell priming, which occurs in lymph nodes. Bone marrow is vascularized by blood but not by lymphatic vessels. Bone marrow is a part of the lymphocyte recirculation network. Billions of lymphocytes circulate through bone marrow each day
(8)
. We found large numbers of functional Tregs in bone marrow. This finding provides evidence for a previously unidentified role of bone marrow in T-cell homeostasis: Bone marrow is a preferential site for migration or selective retention and function of Tregs. The study significantly complements recent reports that bone marrow harbors antigen-specific memory T cells
(20)
and is an important site for T-cell priming
(19)
.

Conventional lymphocyte trafficking has been extensively investigated
(27)
. However, little is known regarding the natural reservoirs for migrating Tregs and the trafficking signals for human Tregs. Our study is the first to show a mechanism for human Treg homeostatic trafficking in vivo.

We also suggest that bone marrow is a significant reservoir for human Tregs and that CXCL12/CXCR4 signals are critical for Treg trafficking between bone marrow and periphery. Several lines of evidence support this notion. First, CXCL12 is expressed in marrow and induces Treg chemotaxis and adhesion/transmigration. Second, G-CSF mobilizes human bone marrow Tregs through reducing bone marrow–derived CXCL12. Third, human Treg preferentially home to bone marrow but not to spleen under homeostatic conditions in NOD/SCID mice. Finally, blocking CXCL12/CXCR4 signals significantly reduces Treg trafficking to bone marrow. There is one amino acid difference between mouse and human CXCL12
(28, 29)
. Thus, our work also suggests that this human–NOD/SCID chimeric model is relevant to study the role of CXCL12/CXCR4 signals in human Treg trafficking.

Expression of FOXP3 can be induced by activation and is associated with the suppressive capacity of Tregs
(22)
. Activated Tregs are more efficient in blocking T-cell activation than nonactivated Tregs
(30)
. Here we show that bone marrow Tregs express more FOXP3 and CD25 than blood counterparts and are functionally superior to their blood counterparts in suppression. We also show that MDC activation of blood Tregs augments their CXCR4 expression, which significantly enhances their migration toward bone marrow–derived CXCL12. Thus, we suggest that bone marrow Tregs contain “memory” and/or activated Tregs.

We showed that human ovarian tumors produce high-level CXCL12 that mediates tumor-associated plasmacytoid dendritic cell trafficking
(15)
. Although we recently showed that there are significant numbers of functional, tumor-infiltrating Tregs in human ovarian tumors, tumor environmental CCL22 but not CXCL12 is crucial for this Treg tumor trafficking in vivo(7)
. Thus, local tumor microenvironmental factors may account for these migratory differences. Alternatively, there may be distinct Treg subsets specifically recruited in response to selected stimuli. In either regard, our data suggest that Treg distribution and trafficking may be regulated in a tissue- and/or organ-specific manner that is further subject to modification by local environmental factors.

Administration of anti-CXCL12 counteracts B1a-lymphocyte expansion and T-lymphocyte activation and decreases autoantibody production and nephritis in murine lupus
(32)
. The CXCR4 antagonist AMD3100 also inhibits autoimmune collagen-induced arthritis in mice
(33)
. These findings may result from Treg mobilization through interrupting CXCR4/CXCL12 signals in bone marrow and suggest additional means for Treg mobilization.

Our data show that recruitment of Tregs into bone marrow through CXCL12 represents a novel and important mechanism of Treg homeostatic traffic. Mobilizing bone marrow Tregs may be a novel strategy to manipulate systemic immunity therapeutically.

Acknowledgments

We thank Dominique Emilie for critical reading of the manuscript, Roy Weiner and Jules Puschett for their support, and Sherry Price for her technical assistance.

Footnotes

Grant support: The Department of Defense (OC020173) and National Cancer Institute (CA092562 and CA100227 to W. Zou and CA100425 to T. J. Curiel).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.